The focus of our research program remains the development of novel positron radiolabeled compounds for PET imaging. Because of the multidisciplinary nature of PET research, all of our research efforts derive from the interests of other scientists within the NIH. The development and evaluation of new potential imaging agents has focused on three areas, all of which are related to the central stress response system. We are exploring ligands for the serotonergic system, the muscarinic acetylcholine system, and the corticotrophin releasing hormone receptor.[unreadable] [unreadable] Our high affinity 5HT1A antagonist, FCWAY, has reached maturity and under a CC/PET department IND is available to all clinical PET research at the NIH. We are preparing a manuscript in collaboration with the NIH/CC describing the automated synthesis of this molecule. We are still investigating other analogues of FCWAY in a search for a compound of somewhat lower affinity that is sensitive to endogenous 5HT. We published a description of our studies in rats to evaluate the effect on binding of our lower affinity candidate, FPWAY, by paroxetine and fenfluramine. We determined that the blood flow changes caused by the administered drug and by anesthesia methods confounded the analysis. The interpretation was that FPWAY was not sensitive to the 5HT releasing effects of the administered drugs in either anesthetized or awake rats. We have also begun a PET imaging study in a monkey model of stress in collaboration with NIAAA investigators. The imaging studies were conducted at NIDA in Baltimore. The first set of studies were completed this year; data analysis is ongoing. [unreadable] [unreadable] Other analogues of FCWAY and FPWAY are being investigated to identify compounds of lower affinity that can be evaluated for 5HT sensitivity and for compounds with lower propensity to give F-18 fluoride as a metabolite. Metabolism was evaluated using metabolites derived from hepatocytes and structural assignments based on mass spectrometry. A manuscript has been submitted that shows that the configuration of the fluorocyclohexane influences the propensity for metabolic defluorination. [unreadable] [unreadable] In the field of muscarinic receptor research, FP-TZTP, our M2 selective agonist, continues to be studied in clinical protocols under IND in the CC/PET Department. Manuscripts that were in press in FY2005 appeared in print this year. We conducted the radiosynthesis of a fluorinated analogue of a M1 selective agonist. The fluorinated compound has proven to be non-selective as demonstrated by in vivo autoradiography studies in M1, M2, M3, or M4 receptor knock out mice. These mice were obtained through collaboration with Jurgen Wess (NIDDK). Evaluation of the tritium labeled non-fluorinated compound showed similar results. Both the fluorinated compound and the tritiated compound demonstrated specific binding, and both showed an M2 component to the binding. However, the selectivity is insufficient to be effective as an imaging agent. A manuscript is in preparation to describe this negative result.[unreadable] [unreadable] Corticotrophin releasing hormone receptors are becoming a major target of imaging agents. In a continuing collaboration with Kenner Rice (NIDDK) and Carlo Contoreggi (NIDA), we have prepared Br-76 labeled ligands for this receptor. We have discovered some interesting chemical anomalies during the course of this synthesis that require explanation. The regiochemistry of the product is dependent on the reaction conditions. By selection of the conditions we can selectively prepare two different isomers of the radiolabeled analog. The different isomers display unique binding profiles in in vitro tissue autoradiography (manuscript in preparation). In vivo studies are troubled with high lipophilicity and low receptor concentrations. [unreadable] [unreadable] New applications of PET radionuclides. We are beginning collaborative investigations with other researchers in the NIH intramural program that will require the development of new radiolabeling strategies. We have explored small fluorinated molecules for selective labeling of proteins on free sulfhydryls and soon plan to study one such protein in a biological system in collaboration with NCI. We also have evaluated a short-chain monofluorinated sulfonyl chloride as a prosthetic group for attachment to primary and secondary amines. Another project has exploited our knowledge in attaching 4-fluorobenzoyl moieties to primary amines on molecules with the potential for studying protein-protein interactions.[unreadable] [unreadable] We have begun a project to radiosynthesize two stereoisomers of a purine-based reverse transcriptase inhibitor. This project will focus on the ability to study pharmacokinetics of therapeutic drugs.